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Sanger Sequencing - Wikipedia
Sanger Sequencing - Wikipedia
Sanger sequencing is a method of DNA sequencing that involves electrophoresis and is based
on the random incorporation of chain-terminating dideoxynucleotides by DNA polymerase during
in vitro DNA replication. After first being developed by Frederick Sanger and colleagues in 1977,
it became the most widely used sequencing method for approximately 40 years. It was first
commercialized by Applied Biosystems in 1986. More recently, higher volume Sanger
sequencing has been replaced by next generation sequencing methods, especially for large-
scale, automated genome analyses. However, the Sanger method remains in wide use for
smaller-scale projects and for validation of deep sequencing results. It still has the advantage
over short-read sequencing technologies (like Illumina) in that it can produce DNA sequence
reads of > 500 nucleotides and maintains a very low error rate with accuracies around 99.99%.[1]
Sanger sequencing is still actively being used in efforts for public health initiatives such as
sequencing the spike protein from SARS-CoV-2[2] as well as for the surveillance of norovirus
outbreaks through the Center for Disease Control and Prevention's (CDC) CaliciNet surveillance
network.[3]
The Sanger (chain-termination) method for DNA sequencing.
Method
The classical chain-termination method requires a single-stranded DNA template, a DNA primer,
a DNA polymerase, normal deoxynucleotide triphosphates (dNTPs), and modified di-
deoxynucleotide triphosphates (ddNTPs), the latter of which terminate DNA strand elongation.
These chain-terminating nucleotides lack a 3'-OH group required for the formation of a
phosphodiester bond between two nucleotides, causing DNA polymerase to cease extension of
DNA when a modified ddNTP is incorporated. The ddNTPs may be radioactively or fluorescently
labelled for detection in automated sequencing machines.
The DNA sample is divided into four separate sequencing reactions, containing all four of the
standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. To each
reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP),
while the other added nucleotides are ordinary ones. The deoxynucleotide concentration should
be approximately 100-fold higher than that of the corresponding dideoxynucleotide (e.g. 0.5mM
dTTP : 0.005mM ddTTP) to allow enough fragments to be produced while still transcribing the
complete sequence (but the concentration of ddNTP also depends on the desired length of
sequence).[4] Putting it in a more sensible order, four separate reactions are needed in this
process to test all four ddNTPs. Following rounds of template DNA extension from the bound
primer, the resulting DNA fragments are heat denatured and separated by size using gel
electrophoresis. In the original publication of 1977,[4] the formation of base-paired loops of
ssDNA was a cause of serious difficulty in resolving bands at some locations. This is frequently
performed using a denaturing polyacrylamide-urea gel with each of the four reactions run in one
of four individual lanes (lanes A, T, G, C). The DNA bands may then be visualized by
autoradiography or UV light, and the DNA sequence can be directly read off the X-ray film or gel
image.
Part of a radioactively
labelled sequencing gel
In the image on the right, X-ray film was exposed to the gel, and the dark bands correspond to
DNA fragments of different lengths. A dark band in a lane indicates a DNA fragment that is the
result of chain termination after incorporation of a dideoxynucleotide (ddATP, ddGTP, ddCTP, or
ddTTP). The relative positions of the different bands among the four lanes, from bottom to top,
are then used to read the DNA sequence.
DNA fragments are labelled with a
radioactive or fluorescent tag on the
primer (1), in the new DNA strand with
a labeled dNTP, or with a labeled
ddNTP.
Chain-termination methods have greatly simplified DNA sequencing. For example, chain-
termination-based kits are commercially available that contain the reagents needed for
sequencing, pre-aliquoted and ready to use. Limitations include non-specific binding of the
primer to the DNA, affecting accurate read-out of the DNA sequence, and DNA secondary
structures affecting the fidelity of the sequence.
Dye-terminator sequencing
Capillary electrophoresis
Dye-terminator sequencing utilizes labelling of the chain terminator ddNTPs, which permits
sequencing in a single reaction rather than four reactions as in the labelled-primer method. In
dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with
fluorescent dyes, each of which emits light at different wavelengths.
Owing to its greater expediency and speed, dye-terminator sequencing is now the mainstay in
automated sequencing. Its limitations include dye effects due to differences in the incorporation
of the dye-labelled chain terminators into the DNA fragment, resulting in unequal peak heights
and shapes in the electronic DNA sequence trace chromatogram after capillary electrophoresis
(see figure to the left).
This problem has been addressed with the use of modified DNA polymerase enzyme systems
and dyes that minimize incorporation variability, as well as methods for eliminating "dye blobs".
The dye-terminator sequencing method, along with automated high-throughput DNA sequence
analyzers, was used for the vast majority of sequencing projects until the introduction of next
generation sequencing.
Applications of dye-terminating
sequencing
The field of public health plays many roles to support patient diagnostics as well as
environmental surveillance of potential toxic substances and circulating biological pathogens.
Public health laboratories (PHL) and other laboratories around the world have played a pivotal
role in providing rapid sequencing data for the surveillance of the virus SARS-CoV-2, causative
agent for COVID-19, during the pandemic that was declared a public health emergency on
January 30, 2020.[8] Laboratories were tasked with the rapid implementation of sequencing
methods and asked to provide accurate data to assist in the decision-making models for the
development of policies to mitigate spread of the virus. Many laboratories resorted to next
generation sequencing methodologies while others supported efforts with Sanger sequencing.
The sequencing efforts of SARS-CoV-2 are many, while most laboratories implemented whole
genome sequencing of the virus, others have opted to sequence very specific genes of the virus
such as the S-gene, encoding the information needed to produce the spike protein. The high
mutation rate of SARS-CoV-2 leads to genetic differences within the S-gene and these
differences have played a role in the infectivity of the virus.[9] Sanger sequencing of the S-gene
provides a quick, accurate, and more affordable method to retrieving the genetic code.
Laboratories in lower income countries may not have the capabilities to implement expensive
applications such as next generation sequencing, so Sanger methods may prevail in supporting
the generation of sequencing data for surveillance of variants.
Sanger sequencing is also the "gold standard" for norovirus surveillance methods for the Center
for Disease Control and Prevention's (CDC) CaliciNet network. CalciNet is an outbreak
surveillance network that was established in March 2009. The goal of the network is to collect
sequencing data of circulating noroviruses in the United States and activate downstream action
to determine the source of infection to mitigate the spread of the virus. The CalciNet network
has identified many infections as foodborne illnesses.[3] This data can then be published and
used to develop recommendations for future action to prevent tainting food. The methods
employed for detection of norovirus involve targeted amplification of specific areas of the
genome. The amplicons are then sequenced using dye-terminating Sanger sequencing and the
chromatograms and sequences generated are analyzed with a software package developed in
BioNumerics. Sequences are tracked and strain relatedness is studied to infer epidemiological
relevance.
Challenges
Common challenges of DNA sequencing with the Sanger method include poor quality in the first
15-40 bases of the sequence due to primer binding and deteriorating quality of sequencing
traces after 700-900 bases. Base calling software such as Phred typically provides an estimate
of quality to aid in trimming of low-quality regions of sequences.[10][11]
In cases where DNA fragments are cloned before sequencing, the resulting sequence may
contain parts of the cloning vector. In contrast, PCR-based cloning and next-generation
sequencing technologies based on pyrosequencing often avoid using cloning vectors. Recently,
one-step Sanger sequencing (combined amplification and sequencing) methods such as
Ampliseq and SeqSharp have been developed that allow rapid sequencing of target genes
without cloning or prior amplification.[12][13]
Current methods can directly sequence only relatively short (300-1000 nucleotides long) DNA
fragments in a single reaction. The main obstacle to sequencing DNA fragments above this size
limit is insufficient power of separation for resolving large DNA fragments that differ in length by
only one nucleotide.
Microfluidic Sanger
sequencing
Microfluidic Sanger sequencing is a lab-on-a-chip application for DNA sequencing, in which the
Sanger sequencing steps (thermal cycling, sample purification, and capillary electrophoresis) are
integrated on a wafer-scale chip using nanoliter-scale sample volumes. This technology
generates long and accurate sequence reads, while obviating many of the significant
shortcomings of the conventional Sanger method (e.g. high consumption of expensive reagents,
reliance on expensive equipment, personnel-intensive manipulations, etc.) by integrating and
automating the Sanger sequencing steps.
In its modern inception, high-throughput genome sequencing involves fragmenting the genome
into small single-stranded pieces, followed by amplification of the fragments by polymerase
chain reaction (PCR). Adopting the Sanger method, each DNA fragment is irreversibly terminated
with the incorporation of a fluorescently labeled dideoxy chain-terminating nucleotide, thereby
producing a DNA “ladder” of fragments that each differ in length by one base and bear a base-
specific fluorescent label at the terminal base. Amplified base ladders are then separated by
capillary array electrophoresis (CAE) with automated, in situ “finish-line” detection of the
fluorescently labeled ssDNA fragments, which provides an ordered sequence of the fragments.
These sequence reads are then computer assembled into overlapping or contiguous sequences
(termed "contigs") which resemble the full genomic sequence once fully assembled.[14]
Sanger methods achieve maximum read lengths of approximately 800 bp (typically 500–600 bp
with non-enriched DNA). The longer read lengths in Sanger methods display significant
advantages over other sequencing methods especially in terms of sequencing repetitive regions
of the genome. A challenge of short-read sequence data is particularly an issue in sequencing
new genomes (de novo) and in sequencing highly rearranged genome segments, typically those
seen of cancer genomes or in regions of chromosomes that exhibit structural variation.[15]
Applications of microfluidic
sequencing technologies
Other useful applications of DNA sequencing include single nucleotide polymorphism (SNP)
detection, single-strand conformation polymorphism (SSCP) heteroduplex analysis, and short
tandem repeat (STR) analysis. Resolving DNA fragments according to differences in size and/or
conformation is the most critical step in studying these features of the genome.[14]
Device design
The sequencing chip has a four-layer construction, consisting of three 100-mm-diameter glass
wafers (on which device elements are microfabricated) and a polydimethylsiloxane (PDMS)
membrane. Reaction chambers and capillary electrophoresis channels are etched between the
top two glass wafers, which are thermally bonded. Three-dimensional channel interconnections
and microvalves are formed by the PDMS and bottom manifold glass wafer.
The device consists of three functional units, each corresponding to the Sanger sequencing
steps. The thermal cycling (TC) unit is a 250-nanoliter reaction chamber with integrated resistive
temperature detector, microvalves, and a surface heater. Movement of reagent between the top
all-glass layer and the lower glass-PDMS layer occurs through 500-μm-diameter via-holes. After
thermal-cycling, the reaction mixture undergoes purification in the capture/purification chamber,
and then is injected into the capillary electrophoresis (CE) chamber. The CE unit consists of a
30-cm capillary which is folded into a compact switchback pattern via 65-μm-wide turns.
Sequencing chemistry
Thermal cycling
In the TC reaction chamber, dye-
terminator sequencing reagent,
template DNA, and primers are loaded
into the TC chamber and thermal-cycled
for 35 cycles ( at 95 °C for 12 seconds
and at 60 °C for 55 seconds).
Purification
The charged reaction mixture
(containing extension fragments,
template DNA, and excess sequencing
reagent) is conducted through a
capture/purification chamber at 30 °C
via a 33-Volts/cm electric field applied
between capture outlet and inlet ports.
The capture gel through which the
sample is driven, consists of 40 μM of
oligonucleotide (complementary to the
primers) covalently bound to a
polyacrylamide matrix. Extension
fragments are immobilized by the gel
matrix, and excess primer, template, free
nucleotides, and salts are eluted through
the capture waste port. The capture gel
is heated to 67-75 °C to release
extension fragments.
Capillary electrophoresis
Extension fragments are injected into
the CE chamber where they are
electrophoresed through a 125-167-
V/cm field.
Platforms
The Apollo 100 platform (Microchip Biotechnologies Inc., Dublin, CA)[16] integrates the first two
Sanger sequencing steps (thermal cycling and purification) in a fully automated system. The
manufacturer claims that samples are ready for capillary electrophoresis within three hours of
the sample and reagents being loaded into the system. The Apollo 100 platform requires sub-
microliter volumes of reagents.
Comparisons to other sequencing
techniques
Performance values for genome sequencing technologies including Sanger methods and next-
generation methods[15][17][18]
Throughput
Injection Average
Number Analysis (including Gel Lane
Technology volume read
of lanes time analysis; pouring tracking
(nL) length
Mb/h)
Capillary array
96 1–5 1–3 hours 700 bp 0.166 No No
electrophoresis
6–30
Microchip 96 0.1–0.5 430 bp 0.660 No No
minutes
Illumina/Solexa 30–100
2–3 days 20
(2008) bp
ABI/SOLiD
8 days 35 bp 5–15
(2008)
Illumina
2x50– 22,000–
NovaSeq 1–2 days
2x150 bp 67,000
(2019)
BGI MGISEQ-
1 day 2x150 bp 250,000
T7 (2019)
Throughput
Injection Average
Number Analysis (including Gel Lane
Technology volume read
of lanes time analysis; pouring tracking
(nL) length
Mb/h)
Pacific
10–20
Biosciences 10–30 kb 1,300
hours
SMRT (2019)
Oxford
13–20
Nanopore 3 days 700
kb[19]
MinIon (2019)
The ultimate goal of high-throughput sequencing is to develop systems that are low-cost, and
extremely efficient at obtaining extended (longer) read lengths. Longer read lengths of each
single electrophoretic separation, substantially reduces the cost associated with de novo DNA
sequencing and the number of templates needed to sequence DNA contigs at a given
redundancy. Microfluidics may allow for faster, cheaper and easier sequence assembly.[14]
See also
Second-generation sequencing
Third-generation sequencing
References
Further reading
External links